Neuromodulators including monoamine neurotransmitters and neural peptides control a wide range of behavioral phenomena, and their dysregulation contributes to neuropsychiatric disease, but we still understand little about the cellular mechanisms that underlie their regulated release. In particular, we do not understand how proteins sort to the large dense core vesicles (LDCVs) that mediate regulated release, rather than to the vesicles that mediate constitutive secretion. Previous work has even suggested a role for lumenal and membrane rather than cytoplasmic interactions of the kind that contribute to other trafficking events such as endocytosis. To identify factors involved in the formation of LDCVs, I am using the vesicular monoamine transporter (VMAT2), a polytopic membrane protein which fills secretory vesicles with monoamine neurotransmitters for exocytotic release. VMAT2 sorts efficiently and directly into the regulated secretory pathway (RSP), and mutations in a conserved, cytoplasmic dileucine-like motif divert VMAT2 to the constitutive pathway, increasing cell surface delivery. This motif appears important for sorting VMAT2 directly into LDCVs, suggesting that, in contrast to previous work, cytoplasmic factors mediate the sorting of VMAT2to the RSP and thus contribute to the biogenesis of LDCVs. We aim to identify the cellular machinery involved in formation of the RSP, using an unbiased RNAi screen in Drosophila S2 cells. Further, we will characterize the role of identified cellular factors in mammalian PC12 cells. In particular, we will determine whether these factors are responsible specifically for the sorting of VMAT2,or more generally for biogenesis of the pathway. Our studies will enable us to identify cellular factors involved in formation of the regulated secretory pathway. This information will allow us to manipulate regulated and constitutive secretion and determine their relative role in normal behavior and psychiatric illness